Tendon tears are common musculoskeletal injuries that heal without restoration of the functional structure. Failure of healing tendons to restore the functional structure leads to progression of injury and high incidence of re-tear after surgical repair. Scarless tendon healing, with restored native tissue properties, could improve surgical outcome or eliminate the need for surgical repair altogether, but is not typically observed in post-natal mammals. The discovery that adult Murphy Roths Large (MRL/MpJ) mice exhibit a regenerative capacity has distinguished this mouse strain as an exciting model to investigate mechanisms that underlie adult regenerative healing. Investigation into the regenerative mechanisms of MRL/MpJ mice has generated hypotheses that implicate both, the systemic and local tissue level, but no conclusive data exists. The parent grant interrogates the contribution of the systemic environment of the MRL/MpJ mouse and the local environment of its tendon to scarless tendon healing. Novel microsurgical techniques and inbred mouse strains are utilized to uncouple the contribution of the systemic environment and the innate tendon to scarless tendon healing. Tendon organ culture is also employed to determine the role of the provisional extracellular matrix (ECM) from regenerative MRL/MpJ mice in modulating the cellular activity that leads to scarless tendon healing. Our findings show that the innate environment of the tendon is essential to the improved healing capacity of the MRL/MpJ tendon. This is evidenced by the fact that organ cultured midsubstance punched MRL/MpJ tendons, wherein the systemic environment is eliminated, exhibit superior recovery of the mechanical properties and the structure than B6 tendons. The parent grant also begins to explore the therapeutic potential of the provisional ECM derived from MRL/MpJ healing tendons to promote scarless tendon healing in scar-mediated B6 mice. More specifically, the provisional ECM from healing MRL/MpJ and B6 tendons will be harvested, decellularized, suspended in a delivery gel and placed in midsubstance punch injured patellar tendons (PT) of scar-mediated B6 mice. The supplement extends the studies proposed in the parent grant by determining the protein and structural composition that are associated with an improved therapeutic outcome. More specifically, the protein composition of the provisional ECM that is associated with the best and worst healing outcomes in B6 mice will be extensively evaluated using proteomics. Furthermore, the structure associated with the optimal composition will be determined from healing MRL/MpJ using Transmission Electron Microscopy (TEM), prior to pulverization. Future studies will use the tools developed in the parent grant (organ culture) and the data from supplement to identify the proteins or network of proteins that promote healing. In addition, the structural data will be used to inform structural design of future delivery scaffolds. In summary, data from the supplement will be instrumental in translating findings from the parent grant into therapeutics.